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Abstract:

Provided are a multiple immunoassay apparatus on a chip in which a
structure comprising multiple microfluidic channels is associated with a
tissue sample, which allows immunohistochemical reactions to be conducted
therein, to examine various markers specific for certain diseases, and a
method for performing multiple immunoassays using the same. The multiple
immunoassay apparatus comprises: at least one antibody-introducing unit
through which at least one antibody is introduced into the apparatus; at
least one reaction unit in which the antibody reacts with a sample in an
immunohistochemical pattern; and at least one fluid outlet through which
a fluid including the antibody is discharged outside the apparatus.

Claims:

1. A multiple immunoassay apparatus, comprising: at least one
antibody-introducing unit through which at least one antibody is
introduced into the apparatus; at least one reaction unit in which the
antibody reacts with a sample in an immunohistochemical pattern; and at
least one fluid outlet through which a fluid including the antibody is
discharged outside the apparatus.

2. The multiple immunoassay apparatus according to claim 1, wherein the
antibody-introducing unit, the reaction unit and the fluid outlet are
interconnected with each other by channels.

3. The multiple immunoassay apparatus according to claim 2, further
comprising a pressure provider for providing pressure to control a flow
rate of fluid within the channels.

4. The multiple immunoassay apparatus according to claim 3, wherein the
pressure provider is a hydraulic or pneumatic device.

5. The multiple immunoassay apparatus according to claim 1, further
comprising at least one fluid controller for controlling fluid flows,
said fluid controller being connected with the antibody-loading unit, the
reaction unit and the fluid outlet via channels.

6. The multiple immunoassay apparatus according to claim 1, further
comprising an electric heater for controlling a temperature of the
apparatus, said electric heater being located on a lower side of the
apparatus to establish a condition optimal for a reaction between an
antibody and a sample.

7. The multiple immunoassay apparatus according to claim 1, wherein there
are as many reaction units as antibody-introducing units.

8. The multiple immunoassay apparatus according to claim 1, further
comprising a transparent cover for covering the apparatus to prevent the
fluid from evaporating.

9. The multiple immunoassay apparatus according to claim 1, further
comprising at least one reagent-introducing unit through which a reagent
necessary for an immunoassay is introduced.

10. The multiple immunoassay apparatus according to claim 1, further
comprising a pressurizing unit for compressing the reaction unit to
promote reactions among the antibody, the reagent and the sample.

11. The multiple immunoassay apparatus according to claim 10, wherein the
pressurizing unit is operated in a bolt-nut manner, a spring compressing
manner, or a balance weight manner.

12. The multiple immunoassay apparatus according to claim 1, wherein the
reaction unit is formed in a zigzag pattern.

13. The multiple immunoassay apparatus according to claim 1, wherein the
antibody and the reagent are reacted with the sample while flowing
through the reaction unit.

14. The multiple immunoassay apparatus according to claim 1, wherein the
sample is a tissue sample or a cell sample.

15. The multiple immunoassay apparatus according to claim 1, having a
lab-on-a-chip structure in which the antibody-introducing unit, the
reagent-loading unit, the reaction unit and the fluid outlet are all
established on a chip.

16. (canceled)

17. A method for performing multiple immunoassays using the multiple
immunoassay apparatus according to claim 1, comprising: loading one or
more antibodies into respective antibody-loading units; providing
pressure to move fluids containing the antibodies to respective reaction
units; allowing the antibodies to react with a sample including a tissue
slice sample to examine markers specific for diseases; and discharging
the fluids through fluid outlets connected to the reaction units.

18. The method according to claim 17, further comprising loading reagents
into respective reagent-introducing units prior to, subsequently to or
simultaneously with the loading of the antibodies.

19. The method according to claim 17, further comprising controlling
flows of the fluids including the antibodies and the reagents to the
sample after the provision of pressure.

20. The method according to claim 17, wherein the pressure is provided
using a hydraulic or pneumatic device.

21. The method according to claim 17, further comprising compressing the
reaction units to promote the reaction between the antibodies and the
sample.

22. The multiple immunoassay apparatus according to claim 9, further
comprising a pressurizing unit for compressing the reaction unit to
promote reactions among the antibody, the reagent and the sample.

23. The multiple immunoassay apparatus according to claim 9, having a
lab-on-a-chip structure in which the antibody-introducing unit, the
reagent-loading unit, the reaction unit and the fluid outlet are all
established on a chip.

24. The method according to claim 18, further comprising controlling
flows of the fluids including the antibodies and the reagents to the
sample after the provision of pressure.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a multiple immunoassay apparatus,
such as fluorescence in situ hybridization of bio-samples including cells
and tissues based on immunohistochemistry. More particularly, the present
invention relates to a multiple immunoassay apparatus in conjunction with
a Lab-on-a-chip technology which is useful in detecting the presence of
enzymes, tumor markers and prognosis markers and determining whether a
tumor is carcinoma or sarcoma, or is benign or malignant, and which can
conduct in a simple manner biological experiments requiring extensive
time and labor.

BACKGROUND ART

[0002] Morphological observations of samples from the lesions of patients
are important for the accurate diagnosis, appropriate treatment, and
study of diseases. Of these, immunohistochemical staining is a highly
sensitive and specific biopsy process which finds a broad spectrum of
applications in the research field related to diagnosis.

[0003] The many advances of the last two decades in the
immunohistochemical staining field have introduced staining methods
capable of quickly reading out results, allow for retrospective studies
with paraffin slides, and have developed thousands of readily usable
antibodies for detecting antigens.

[0004] Immunohistochemistry having such advantages is now recognized as a
fundamental and effective diagnostic method, but requires an examination
of various markers of cancer to make an accurate diagnosis of cancer and
to conduct appropriate chemotherapy and molecular therapy for the
patients. However, limited tissue samples, low yields involved with
preparing paraffin blocks from tissue samples, and economic and time
burdens on patients make it clinically difficult to make an examination
of various markers.

DISCLOSURE

[Technical Problem]

[0005] Accordingly, the present invention has been made keeping in mind
the above problems occurring in the prior art, and an object of the
present invention is to provide a multiple immunoassay apparatus on a
chip in which a structure comprising multiple microfluidic channels is
associated with a tissue sample, which allows immunohistochemical
reactions to be conducted therein, to examine various markers specific
for certain diseases, and to provide a method for performing multiple
immunoassays using the same.

[Technical Solution]

[0006] In order to accomplish the above objects, the present invention
provides a multiple immunoassay apparatus, comprising: at least one
antibody-introducing unit through which at least one antibody is
introduced into the apparatus; at least one reaction unit in which the
antibody reacts with a sample in an immunohistochemical pattern; and at
least one fluid outlet through which a fluid including the antibody is
discharged outside the apparatus.

[0007] In a modification of the apparatus, the antibody-introducing unit,
the reaction unit and the fluid outlet are interconnected with each other
through channels.

[0008] In another modification, the apparatus further comprises a pressure
provider for providing a pressure to control a flow rate of fluid within
the channels.

[0009] In this modification, the pressure provider is a hydraulic or
pneumatic device.

[0010] In a further modification, the apparatus further comprises at least
one fluid controller for controlling fluid flows, the fluid controller
being connected with the antibody-loading unit, the reaction unit and the
fluid outlet via channels.

[0011] In yet a further modification, the apparatus further comprises an
electric heater for controlling a temperature of the apparatus, the
electric heater being located on a lower side of the apparatus to
establish a condition optimal for a reaction between an antibody and a
sample.

[0012] In yet another modification of the apparatus, the number of
reaction units is the same as the number of the antibody-introducing
unit.

[0013] In still another modification, the apparatus further comprises a
transparent cover for covering the apparatus to prevent the fluid from
evaporating.

[0014] In still a further modification, the apparatus further comprises at
least one reagent-introducing unit through which a reagent necessary for
an immunoassay is introduced.

[0015] In still yet another modification, the apparatus further comprises
a pressurizing unit for compressing the reaction unit to promote
reactions between the antibody, the reaction and the sample.

[0016] In this modification, the pressurizing unit is operated in a
bolt-nut manner, in a spring compressing manner, or in a balance weight
manner.

[0017] In still yet a further modification of the apparatus, the reaction
unit is formed in a zigzag pattern.

[0018] In an additional modification of the apparatus, the antibody and
the reagent react with the sample while flowing through the reaction
unit.

[0019] In another additional modification of the apparatus, the sample is
a tissue sample or a cell sample.

[0020] In a yet additional modification, the apparatus has a lab-on-a-chip
structure in which the antibody-introducing unit, the reagent-loading
unit, the reaction unit and the fluid outlet are all established on a
chip.

[0021] In a further additional modification of the apparatus, the reaction
unit has a width of 3 cm or smaller.

[0022] In accordance with another aspect thereof, the present invention
provides a method for performing multiple immunoassays using the
apparatus, comprising: loading one or more antibodies into respective
antibody-loading units; providing a pressure to move fluids of the
antibodies to respective reaction units; allowing the antibodies to react
with a sample including a tissue slice sample to examine markers specific
for diseases; and discharging the fluids through fluid outlets connected
to the reaction units.

[0023] In a modification thereof, the method further comprises loading
reagents to respective reagent-introducing units prior to, subsequently
to or simultaneously with the loading of the antibodies.

[0024] In another modification thereof, the method further comprises
controlling flows of the fluids including the antibodies and the reagents
to the sample after the provision of pressure.

[0025] In a further modification of the method, the pressure is provided
using a hydraulic or pneumatic device.

[0026] In still a further modification thereof, the method further
comprises compressing the reaction units to promote the reaction between
the antibodies and the sample.

ADVANTAGEOUS EFFECTS

[0027] As described above, the apparatus and the method for performing
multiple immunoassays in accordance with the present invention can reduce
the consumption of expensive antibodies to one tenth that of conventional
apparatuses and methods.

[0028] In addition, the apparatus and method of the present invention
allows various markers to be detected on even one tissue slice sample,
thus surmounting the low yield of immunohistochemistry, and can secure
medical information on various tissues of a cancer patient within a short
period of time, ensuring accurate diagnosis leading to optimal chemo- and
molecular therapy.

[0029] Further, the apparatus and method of the present invention can
obtain information on the prognosis of patients which is greatly helpful
in choosing therapeutic methods and reduces biochemical and medical
examinations in terms of time and cost, thus allowing medical treatments
to be rapidly applied to patients with a great reduction in financial
burden of the patients.

DESCRIPTION OF DRAWINGS

[0030] FIGS. 1 and 2 are, respectively, structural and cross-sectional
views of a multiple immunoassay apparatus in accordance with a first
embodiment of the present invention,

[0031] FIG. 3 is a structural view of a multiple immunoassay apparatus in
accordance with a second embodiment of the present invention,

[0032] FIGS. 4 to 6 are cross-sectional views of the multiple immunoassay
apparatus of FIG. 3,

[0033] FIG. 7 is a cross-sectional view of the multiple immunoassay
apparatus in accordance with the second embodiment of the present
invention, covered with a cover,

[0034] FIG. 8 is a cross-sectional view of a multiple immunoassay
apparatus in accordance with a third embodiment of the present invention,

[0035] FIG. 9 is a structural view of a multiple immunoassay apparatus in
accordance with a fourth embodiment of the present invention,

[0036] FIG. 10 is a structural view of a multiple immunoassay apparatus in
accordance with a fifth embodiment of the present invention,

[0037] FIG. 11 is a flow chart showing the setting process of the multiple
immunoassay apparatus.

[0038] FIG. 12 is a flow chart showing a multiple immunoassay method,

[0039] FIG. 13 shows one cellblock sample which is immunoassayed with
various antibodies using the multiple immunoassay apparatus of the
present invention.

BEST MODE

[0040] In accordance with an aspect thereof, the present invention
provides a multiple immunoassay apparatus, comprising: at least one
antibody-introducing unit through which at least one antibody is
introduced into the apparatus; at least one reaction unit in which the
antibody reacts with a sample in an immunohistochemical pattern; and at
least one fluid outlet through which a fluid including the antibody is
discharged outside the apparatus.

[0041] In accordance with another aspect thereof, the present invention
provides a method for performing multiple immunoassays, comprising:
loading one or more antibodies into respective antibody-loading units;
providing pressure to move fluids of the antibodies to respective
reaction units; allowing the antibodies to react with a sample including
a tissue slice sample to examine markers specific for diseases and
discharging the fluids through fluid outlets connected to the reaction
units.

Mode for Invention

[0042] Hereinafter, embodiments of the present invention will be described
in detail with reference to the attached drawings.

First Embodiment

[0043] With reference to FIGS. 1 and 2, a multiple immunoassay apparatus
on a chip in accordance with a first embodiment of the present invention
is shown in a structural diagram and a cross sectional view,
respectively. The apparatus according to the first embodiment of the
present invention comprises at least one antibody loading unit, at least
one reaction unit and at least one fluid outlet.

[0044] First, operation and functions of the multiple immunoassay
apparatus will be described, consulting these figures.

[0045] The present invention may be applied to various biological assays,
such as fluorescence in situ hybridization (FISH), and
immunohistochemistry is now delineated as a representative example. As
shown in FIG. 1, the apparatus comprises eight antibody loading units
111˜118, numbered one to eight, through which primary antibodies
necessary for immunohistochemistry are introduced. The processes of the
immunohistochemistry prior to the reaction of the primary antibodies may
be conducted in a typical manner.

[0046] Communicating with the reaction unit via channels, there are as
many antibody loading units as there are channels. Each antibody loading
unit may further comprise a fluid controller for controlling a flow of
antibody by opening or closing the channels. The fluid controller and the
antibody loading unit may be increased or decreased in number if
necessary. In this embodiment, eight antibody loading units are given. A
fluid containing a primary antibody suitable for use in
immunohistochemistry is loaded on the antibody loading unit. In the first
embodiment, 8 reaction unit channels and are provided, and the
corresponding 8 antibody loading units, that is, a first antibody loading
unit 111 to an eighth antibody loading unit 118, are provided as well.

[0047] In addition, the apparatus in accordance with the first embodiment
of the present invention may further comprise a pressure provider,
connected to the channel, for controlling the flow rate of the fluid in
the channel. As the pressure provider, a pneumatic or hydraulic device,
such as a syringe pump, may be used. As the pressure provider controls
the flow rates within the channel, the antibody is allowed to effectively
react with the tissue sample. As for the driving force behind the fluid
flow, it may be obtained from the pump connected with a fluid outlet 151
or may be the result of direct pressure applied to the antibody loading
unit. Alternatively, an antibody alone may be loaded to the
antibody-loading unit so as to react with a sample present underneath it,
without applying direct pressure to the antibody-loading unit.

[0048] In the multiple immunoassay apparatus in accordance with a first
embodiment of the present invention, as described above, antibodies are
loaded onto the antibody-loading units 111˜118 and flow into the
reaction units 131˜138 under the cooperative control of the
pressure provider and the fluid controller where they are subjected to
multiple immune reactions with a sample 141 including tissues samples,
cells and the like, followed by discharge via the fluid outlet 151.
Preferably, each of the reaction units is 3 cm or smaller in width
because widths of tissue samples generally do not exceed 3 cm and many
reaction units are provided for multiple immunoassays. Further, the
multiple immunoassay apparatus in accordance with the present invention
may be prepared in the structure of a lab-on-a-chip, ensuring the simple
and effective implementation of immunoassays.

[0049] The structure and assemblage of the multiple immunoassay apparatus
in accordance with the present invention is useful in understanding the
operation thereof. With reference to FIG. 2, a sample 141 placed on a
lower assembly plate 200 is assembled with the apparatus of FIG. 1 which
is associated with a compression plate 400 and an upper assembly plate
500. Here, the apparatus is preferably assembled with the sample in such
a manner that a fluid, such as a blocking solution, is positioned on the
sample. When the apparatus is correctly assembled, the first to the
eighth antibodies are introduced through the antibody loading units
111˜118, respectively. Although eight antibody loading units are
provided in this embodiment, the number may vary depending on
requirements or designs. In the reaction units 131˜138 which are
respectively connected via channels to the antibody loading units,
antibodies, after migrating thereto via the channels, interact with
tissue samples. A pressurizing unit 600 is provided for pressurizing the
reaction units to effectively carry the antibody to the reaction units,
thus leading to sufficient reaction between the reagents, the antibodies
and the samples. The pressurizing unit 600 is operated using a bolt-nut
joint, a balance weight, or a spring compressor.

[0050] Also, the apparatus may further comprise a pressure provider,
connected to both the antibody loading unit and the reaction unit, for
controlling the flow rate of the fluid within the channel. As an example
of the pressure provider, a syringe pump is connected to the fluid outlet
151, generating a vacuum to allow the antibodies to flow toward the
reaction units and to react with samples in the reaction units. After
completion of the reaction, the pressurizing unit is loosened to separate
the compression plate and the upper assembly plate from the sample. Then,
the sample may undergo typical processing following the primary antibody
reaction.

[0051] Through the fluid outlet 151, fluids including antibodies which
remain unbound to the sample are discharged. As described above, a
pressure provider, such as a device for generating a vacuum, like a
syringe pump, may be applied to the fluid outlet to help the discharge of
the fluid.

[0052] In addition, the apparatus may further comprise an electric heater
for controlling a temperature to promote the reaction. Located on the
bottom surface of the apparatus, the electric heater functions to
maintain a temperature at which optimal reactivity can occur. For
fluorescence in situ hybridization (FISH), the electric heater can
achieve a suitable temperature. Also, cells may be cultured in a vessel
adaptable to the channel of the reaction unit. Thus, the researcher can
conducted desired molecular biological or cell biological experiments
without limitations as to the apparatus. Grooves, although not shown, are
formed at the verge of a sample support on the lower plate 200 to contain
liquid such as water therein, thereby preventing the evaporation of the
reagents and antibodies introduced into the multiple immunoassay
apparatus. This can reduce the consumption of expensive antibodies.

Second Embodiment

[0053] Referring to FIG. 3, a multiple immunoassay apparatus on a chip in
accordance with a second embodiment of the present invention is shown. As
shown, the multiple immunoassay apparatus on a chip in accordance with
the second embodiment of the present invention further comprises a
reagent introducing unit through which a reagent necessary for
immunohistochemistry is introduced into the apparatus. In this example, a
first reagent introducing unit 101 to a sixth reagent introducing unit
106 are provided. For instance, the first reagent introducing unit 101
may be provided for TBS Tween washing buffer, the second reagent
introducing unit 102 for a biotinylated secondary antibody, the third
introducing reagent unit 103 for streptavidin-HRP, the fourth reagent
introducing unit 104 for distilled water, the fifth reagent introducing
unit 105 for DAB, and the sixth reagent introducing unit 106 for Mayer's
hematoxylin. The number of reagent introducing units may be increased or
decreased according to the number of required reagents.

[0054] Various reagents necessary for biological experimentation, such as
those necessary for immunohistochemistry, can be, thus, readily
introduced through the reagent introducing units of the present
invention. The reagent introducing units are connected with respective
fluid controllers 121˜126 for controlling flow rates of the
reagents.

[0055] There are as many antibody loading units, communicating with both
the reaction units and the reagent introducing units, as there are
channels of the reaction units. Each of the antibody loading units is
also provided with a fluid controller 128 for controlling the flow rate
of the antibodies. The antibody loading units may vary in number as well,
depending on requirements. In the same manner as in the First Embodiment,
fluids in the reagent introducing units, the antibody loading units and
the channels are controlled by the fluid controllers 121˜129. That
is, the fluid controllers are connected to the reagent introducing units,
the antibody loading units, the reaction units and the fluid outlet,
respectively, and control the flow rates of many fluids.

[0056] As in the first Embodiment, the apparatus comprises a pressure
provider, connected to the channel, for providing pressure to control the
flow rate of the fluid within the channel. As the pressure provider, a
pneumatic or hydraulic device, such as a syringe pump, may be used. Under
the control of flow rates within the channel by the pressure provider,
the antibody is allowed to effectively react with the tissue sample. As
for the driving force for the fluid flow, it may be obtained from the
pump connected with a fluid outlet 151 or may result from direct pressure
applied to the antibody loading unit. The number of the fluid controllers
connected via valves respectively to the fluid channels may be increased
or decreased depending on the number of both the reagent introducing
units and the antibody loading units.

[0057] In the reaction units which are respectively connected via the
channels to the antibody loading units, antibodies, after migrating
thereto via the channels, interact with tissue samples. A pressurizing
unit 600 is provided for pressurizing the reaction units to effectively
carry the antibody to the reaction units, thus leading to sufficient
reaction between the reagents, the antibodies and the samples. The
pressurizing unit 600 is operated using a bolt-nut joint, a balance
weight, or a spring compressor.

[0058] The tissue samples reactive to the antibodies include tissues and
cells. For the convenience of binding between the antibodies and the
tissue samples, an assembler may be provided. Different reagents may be
introduced into the reaction units via the channels, allowing the
simultaneous implementation of various experiments.

[0059] Through the fluid outlet, fluids including reagents and unbound
antibodies are discharged. The fluid outlet may be connected with a
pressure provider via a tube. In this regard, the tube may be applied to
the fluid outlet device at any time during the above-mentioned procedure.
A representative example of the pressure provider includes a syringe
pump.

[0060] An application of a cover 900 to the apparatus according to the
Second Embodiment is shown in FIG. 7. The cover, made of a transparent
material, prevents the evaporation of the fluids used in the apparatus,
allowing the view of the immune responses therethrough. In addition to
the cover 900, the sample support 300, the compression plate 400 and the
upper assembly plate are preferably made of transparent materials for the
convenience of microscopic observation. Also, the upper assembly plate
has openings for channels of the multiple immunoassay apparatus, for
example, at positions for the reagent introducing units, the fluid
controllers, the antibody loading units and the fluid outlet. No
explanations are given of the other constituents because they are the
same as in the Second Embodiment.

Third Embodiment

[0061] FIG. 8 is a cross sectional view of a multiple immunoassay
apparatus in accordance with a third embodiment of the present invention.
This apparatus, as described above, comprises a compression
plate-incorporated upper assembly plate 450 having openings for the
reagent introducing units, the fluid controllers, the antibody loading
units and the fluid outlet, which is arranged with the lower assembly
plate. In this embodiment, the upper assembly plate and the compression
plate are integrated into one plate so that the apparatus is more simple
in organization and can be more easily assembled.

Fourth Embodiment

[0062] FIG. 9 shows an apparatus according to a fourth embodiment of the
present invention. In contrast to the straight reaction units of FIGS. 1
and 2, the reaction units of this embodiment are in zigzag form which
ensures larger areas for the reaction between the samples and the
antibodies to improve the reliability of the immunoassays. Although the
reaction units are shown in the form of zigzags symmetric with respect to
the center, it should be understood that various forms may be applied to
the entirety of reaction units in order to achieve the above-mentioned
intention, that is, to increase the area of the entire reaction units.
The apparatus of FIG. 11 is identical in operation and order to that of
FIG. 1 or 2.

Fifth Embodiment

[0063] FIG. 10 shows an application of the multiple immunoassay apparatus
of the present invention to a tissue microarray 541. In this embodiment,
as shown in FIG. 10, tissue samples are arranged in the form of an array
on a slide glass 543 and are overlaid with the multiple immunoassay
apparatus to give a system in which various tissue samples can be
monitored for immune responses to various antibodies. In this figure, the
arrayed tissue samples are depicted as smaller than the channels of the
reaction units of the multiple immunoassay apparatus. However, if the
tissue samples are prepared in a size larger than the total width of the
reaction units, each of various tissue samples can be assayed for
reactions with various antibodies. In order to achieve this, the reaction
unit channels of the multiple immunoassay apparatus in accordance with
the present invention must be fabricated into an array form which is
lined with the tissue samples placed on the slide glass. It should be
noted that this organization is included within the scope of the present
invention.

[0064] Next, a brief explanation is given of the assay method using the
multiple immunoassay apparatus. First, a tissue slide to be inspected is
placed on a slide glass, followed by positioning the multiple immunoassay
apparatus of FIG. 1 or 3 thereover. In this regard, the reaction units of
the multiple immunoassay apparatus are aligned with the tissue sample at
the position 141 to be inspected. It is very important to correctly align
the reaction units with the sample located underneath. After the
alignment, reagents and antibodies are loaded into the reagent
introducing units and antibody loading units respectively. A pump
connected to the fluid outlet is operated to flow the fluids at suitable
flow rates, for example, at a flow rate of 50 μl/h.

[0065] The application of the apparatus of the present invention to
immunohistochemistry enjoys the advantage of obtaining information on the
reaction of various antibodies with one tissue sample. If the fluid
controller 128 is open while the other valves 121˜127 and 129 are
closed, four different antibodies 111˜114 are allowed to flow
towards the four reaction channels 131˜134, respectively and react
with a sample in the respective reaction units. In order to maintain the
reaction of the sample with the antibodies, they may be allowed to slowly
flow or may be confined within the reaction units by closing the fluid
controller 129.

[0066] After completion of the immune responses, reagents may be allowed
to flow to the reaction units according to immunohistochemical
procedures. For example, when the valves 121, 127 and 129 are open while
the other valves 122, 123, 124, 125, 126 and 128 are closed, TBS Tween
washing buffer loaded to the first reagent introducing unit flows through
the four reaction channels. In a manner similar to this, the other
reagents can be allowed to flow through the channels. As each channel is
being used it may be observed under a microscope to examine the reaction
of the sample. The images thus obtained may be analyzed in a subsequent
process. Alternatively, the sample support is separated from the multiple
immunoassay apparatus after the completion of the reaction, and observed
under a microscope. Also, the sample support may be stored separately.
The above-described setting and examination processes of the apparatus
are summarized in the flow charts of FIGS. 11 and 12.

[0067] FIG. 11 is a flow chart showing the setting process of the multiple
immunoassay apparatus.

[0068] First, a lower assembly plate is prepared. A sample support is
placed on the lower assembly plate (S401). The multiple immunoassay
apparatus, after being positioned over the sample support, is treated
with plasma (S402) and distilled water is dropped on the lower assembly
plate and the sample support (S403) before the alignment of the apparatus
with the sample support (S404). Thereafter, an examination is made of the
occurrence of bubbles between the sample support and the apparatus
(S405). When bubbles are formed between the sample support and the
apparatus, they are disassembled (S406). Then, the multiple immunoassay
apparatus is aligned with the sample support lest bubbles should be
formed (S404). Upon the reformation of bubbles, the realignment of the
sample support with the apparatus is repeated until the bubbles are not
formed.

[0069] When no bubbles are formed after the alignment of the sample
support with the multiple immunoassay apparatus, an upper assembly plate
is laid on the apparatus and fixed to the lower assembly plate with a
pressurizing unit for compressing the apparatus (S407). After the
compression of the tissue sample and the multiple immunoassay apparatus,
reagents and antibodies are loaded into the reagent introducing units and
the antibody loading units, respectively (S408). In order to allow the
reagents and the antibodies to flow well, the fluid controllers
communicating with the reagent introducing units, the antibody loading
units and the fluid outlet are connected with each other (S409). The
fluid outlet, extending through channels to the reaction units, is
connected via a tube to a pump for generating a vacuum to promote the
flow of the fluids through the reaction units (S410).

[0070] Subsequently, an examination is made of whether the fluids are
flowing well through channels (S411). If the fluids are not flowing or
the fluid controllers are not operated appropriately, the pressurizing
unit for compressing the apparatus and the tissue sample is controlled
such that the fluids flow well or that the fluid controllers are operated
well (S412). Therefore, if the fluids flow well and the fluid controllers
function well, basic settings are said to be established.

[0071] With reference to FIG. 12, an immunoassay method is illustrated in
a flow chart.

[0072] First, at least one reagent and at least one antibody are loaded
into the reagent introducing unit and the antibody loading unit,
respectively (S501). Next, the following measures are taken, before
reactions with tissue samples, in order to establish optimal conditions
for the reactions.

[0073] Valves 127 and 129 for the fluid controllers containing a
deparaffinized solution are opened after which a 100% ethanol valve, a
95% ethanol valve, a 80% ethanol valve, a 70% ethanol valve and a
distilled water valve are opened in that order, to conduct the hydration
of the tissue (S502). While the electric heater located at a lower side
of the sample support supporting the apparatus is set at 90° C., a
valve containing citrate buffer or TRIS-EDTA buffer is opened. The
electric heater is turned off to keep the temperature of the reaction
unit at room temperature, which is optimal for reactions between the
antibodies and the tissue sample (S503).

[0074] After an optimal condition for the reactions is maintained, the
presence of bubbles inside the channels is examined (S504). Bubbles
within the channels inhibit the reactions. When bubbles are observed
within the channels, all of the open valves are closed and the bubbles
are removed from the channel by applying a pressure through the pump
connected to the fluid outlet via the tube (S505). A representative
example of this pump includes a syringe pump. This procedure is repeated
until no bubbles are being formed.

[0075] If no bubbles are present inside the channels, a hydrogen peroxide
valve, a TBS Tween buffer valve, and a Blocking solution valve are opened
sequentially in that order while the fluid controller valve 127 is
closed.

[0076] After the completion of the washing and temperature control, a
pressure is applied so as to move the reagents and the antibodies to the
reaction units (S506). If the antibodies are not completely moved to the
reaction units, a vacuum is applied through the pump to fill the reaction
units with the antibodies.

[0077] When moved to the reaction units, the antibodies react with the
tissue sample so that various markers can be detected (S507). At this
time, the reaction units may be pressurized to promote this reaction
(S508). After completion of the reaction, a TBS Tween buffer valve, a
Streptavidin HRP valve, and a distilled water valve are sequentially
opened in that order, followed by opening a DAB valve, a distilled water
valve and a Mayer's Hematoxylin valve and then a distilled water valve,
an ethanol valve and a Xyline valve. In this state, microscopic images
are obtained and analyzed. Thereafter, the fluids including the reagents
and antibodies are discharged through the fluid outlet connected to the
reaction units (S509). Through these processes, the multiple immunoassays
are completed.

[0078] FIG. 13 shows one cellblock sample which is immunoassayed with
various antibodies using the multiple immunoassay apparatus of the
present invention. FIG. 13A is a view of a sample (breast cancer cell
line: AU-565) on which the multiple immunoassay apparatus is placed.
Immunohistochemical results of the sample with respect to antibodies to
estrogen receptor, HER2, progesterone receptor and Ki67 are shown in FIG.
13B. As seen in FIG. 13C, responses to various antibodies can be achieved
on one sample with distinct immunohistochemical results.

[0079] Although the preferred embodiments of the present invention have
been disclosed for illustrative purposes, those skilled in the art will
appreciate that various modifications, additions and substitutions are
possible, without departing from the scope and spirit of the invention as
disclosed in the accompanying claims.

INDUSTRIAL APPLICABILITY

[0080] Capable of obtaining extensive information on the prognosis of
patients, the apparatus and method of the present invention is greatly
helpful in choosing therapeutic methods and reduces biochemical and
medical examinations in terms of time and cost, thus allowing medical
treatments to be rapidly applied to patients with a great reduction in
financial burden of the patients.

Patent applications by Eun Sook Lee, Seoul KR

Patent applications by Je-Kyun Park, Daejeon KR

Patent applications by KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY